Study On The Preparation Of Staphylococcal Enterotoxin C2

Staphylococcal enterotoxins(SEs) are series of exotoxins secreted by Staphylococcus aureus, they are also typical superantigen(SAg),which could stimulate a large number of T cell with an extreme low concentration. With this characteristic, SEs and other SAg have a wild prospect of application in anti-tumor field. At present, about 20 kinds of SEs were found, include SEA~SEE(the SEC include SEC1,SEC2 and SEC3), SEG~SEQ and TSST-1, and more of them are being explored. Though the SEs have reasonable theoretics of anti-tumor effect, there is few drug was developed based on it, because there are still a lot of troubles in the application of pure SEs on patients. In our country, a drug called Jinpuye Preparation, made from S. aureus broth, was used in assistant clinical therapy for some malignancy tumors. Because trace SEC was claimed to be detected in Jinpuye Preparation by Shenyang Xiehe Group, they made SEC as the criterion for the effective element of this drug. But the concentration of SEC in Jinpuye Preparation is so low, and there are also many other metabolites co-existed with it, moreover, Jinpuye Preparation have some positive curative effect that could not be explained by SAg mechanism, it's hard to draw the conclusion that the SEC is the only active element of the drug.To obtain pure SEC and lay the foundation of deep research on SEC anti-tumor mechanism, SEC2, which is one of antigenic variants of SEC, was chosen as the target protein. Firstly, native SEC2(nSEC2) was purified by anion-exchange chromatography and molecular exclusion chromatography from S. aureus broth, after pretreated with ultra-filtration and sulfate ammonium precipitation. The purity of nSEC2 was about 90%, but the amount is not adequant. Because our laboratory have excellent experience in recombinant protein expression, secondly, sec2 gene was cloned from S. aureus genome and expressed in E. coli, the rSEC2 was purified by anion-exchange chromatography and molecular exclu-sive chromatography, resulted in more than 90% of purity and higher productivity compare with nSEC2. Tag protein with polyhistidine is a favorite strategy for recombinant protein expression and purification, to improve the purity of target protein, thirdly, sec2 gene was cloned and the rSEC2(named as rSEC2-His) was expressed in E. coli with a His-Tag on it's C-terminal, the protein was purified by affinity chromatography. MTT method was applied in testing the bioactivity of the three proteins. The SEC2(include nSEC2 and two rSEC2) is easily ruptured during purification and store, on SDS-PAGE, it exhibit to be two peptides with molecular weight of 13kDa and 15kDa respectively. The 3D structure of SEC2 also shows that there is a nick on the disulphide loop. The effect of a series of factors on this phenomenon were checked, and the mechanism of this phenomenon was also analyzed. 1. Preparation of nSEC2The standard strain for nSEC2 production is S. aureus FRI 1230. The fermentation condition optimized by Uniform Design, and a culture condition was decided: 0.8% Tryptone, 0.6% Yeast Extract, 0.2% Glucose and 0.4% NaCl. Fermentation was carried out at 37°C, after 24h, the broth was separated by centrifugal, the supernatant was further filtrated through 0.45jxm membrane and condensed 5 fold by ultrafiltration.The concentrate was salted out by sulphate ammonium, the deposite collection of 60%~85% saturation with target protein was resuspend with 5mmol/L Tris(pH8.0), and dialysised against 5mmol/L Tris(pH8.0) at 4°C. The crude protein loaded to lOmL DEAE-Sepharose, and eluted with 0.08mol/L NaCl(pH8.0), then the eluate was loaded to 16X40 chromatography column filled with 60mL Sephadex G-75. The elution buffer for molecular exclusion is O.lmol/LNaCl, 5mmol/L Tris(pH8.0). After two step of purification, the target protein was one single band in SDS-PAGE. Purity of the purified nSEC2 was about 90%, test by densitometric scan.The bioactivity of nSEC2 was validated by T-cell stimulation test and tumor cell inhibition test. In the T-cell stimulation test, splenic lymphocyte of ICR mice was used as target cell, culture and Con A were used as control and positive control respectively. The measure wavelength is 570nm, and the reference wavelength is 630nm, the value of 570nm —630nm was set up as reference value. Different concentration of nSEC2 was tested for the proliferation effect on the target cell, and the result showed that low concentration ofnSEC2 could stimulate proliferation of target cell obviously. In tumor cell inhibition test, K562 and K562-AD cell were used as target cell, and the splenic lymphocyte was used as contributing cell. Different concentration of nSEC2 was tested the lethal effect on the target cell through stimulating the contributing cell. The result showed that low concentration of nSEC2 could inhibit the target cell efficiently.2. Expression and purification of recombinant SEC2The sec2 gene was amplified from genome of S. aureus FRI 1230 by standard PCR. Sequencing result of the gene indicated that there were 3 different nucleotides compared with the correspond sequence in GenBank, one of the difference results in the change of amino acid residues. A site-directed mutation kit was applied and the nucleotide was mutated successfully. A pair of primers carry with Nco I and Xho I endonucleases sites respectively was designed, with these primers, sec2 gene with these two sites was amplified by PCR, and cloned into pET-28a(+) plasmid subsequently. The constructed expression vector was named as pET-28a-,rec2,after transformed into competent E. coli BL21 strain, the positive clones were screened by Kanamycin.The chosen expression strain was cultured with 2 X YT culture and induced with 0.5 X 10'3mol/L IPTG Collected E. coli was breaked up by FRENCH? pressure cell press, and ultrasonic was applied to decrease viscosity. The rSEC2, with an excellent solubility, was about 40% in the total protein. After loaded to lOmL DEAE-Sepharose, the rSEC2 was eluted with 0.08mol/L NaCl(pH8.0), then the eluate was loaded to Sephadex G-75 column. The elution buffer for molecular exclusion is identical with that of used in nSEC2 purification. Purity of the purified rSEC2 was more than 90%. The rSEC2 has same bioactivity as nSEC2 through T-cell stimulation test and tumor cell inhibition test.3. Expression and purification of recombinant SEC2 with His-TagA pair of primers was designed for the another strategy: the forward primer contain a Nco I site and start code, the reverse primer contain a Xho I site, stop code and nucleotides code for 6 X His. The sec2 gene with the tag was subcloned from mutated pGEM-sec2, and inserted into expression vector pET-28a(+), the reconstructed expression vector named as pET-28a-sec2(His). The expression host is E. coli BL21, the positive clones were screened by kanamycin, and cultured in 2 X YT culture. The induce condition and proteinpretreatment method is identical with the former.The rSEC2-His was purified by affinity chromatography. The crude protein solution was loaded to HisBind? quick 900 cartridge, then washed by binding buffer and washing buffer, at last, the target with a purity of more than 90% was eluted with elution buffer (lmol/L imidazol). The imidazol in eluate was replace with 5mmol/L Tris by ultrafiltration.West blotting proved that the rSEC2-His and nSEC2 has similar epitopes. T-cell stimulation test and tumor cell inhibition test also the proved the typical superantigen activity of rSEC2-His. 4. Research on the nick of SEC2The purified SEC2 is easily ruptured, on SDS-PAGE, it exhibit to be two peptides with molecular weight of 13kDa and 15kDa respectively, but in nature state, the two peptides are united as one molecule. The 3D structure of SEC2 shows that there is a nick on the disulphide loop. Analyzed the effect of different pH, oxidants, reducing agents, denaturants and protease inhibitors on appearance of the SEC2 nick, a conclusion was drawn that the nick was arosed by some kinds of proteases. Different parts of the crude protein were also tested to identify in which part the protease was existed. The SEC2 was found having the presumable protease activity, and probably relates to the nick, this phenomenon is also probably a self-mature process of the protein.In this research, nSEC2, with purity of about 90%, rSEC2, more than 90%, and rSEC2-His, more than 97%, were obtained, which lay the foundation for the preparation of monoclonal antibody and polyclonal antibody of SEC2. MTT method validated the SAg bioactivity of three SEC2: stimulate splenic lymphocyte proliferation and inhibit tumor cell with low concentration, which lay the foundation for further research on the SEC2 anti-tumor mechanism and identifying the active element in Jinpuye Preparation. Presumable protease activity of SEC2 was firstly observed, which may be concerned with formation of the nick.